Delivery of therapeutic biologicals from implantable tissue matrices

ABSTRACT

Normal cells, such as fibroblasts or other tissue or organ cell types, are genetically engineered to express biologically active, therapeutic agents, such as proteins that are normally produced in small amounts, for example, MIS, or other members of the TGF-beta family Herceptin™, interferons, andanti-angiogenic factors. These cells are seeded into a matrix for implantation into the patient to be treated. Cells may also be engineered to include a lethal gene, so that implanted cells can be destroyed once treatment is completed. Cells can be implanted in a variety of different matrices. In a preferred embodiment, these matrices are implantable and biodegradable over a period of time equal to or less than the expected period of treatment, when cells engraft to form a functional tissue producing the desired biologically active agent. Implantation may be ectopic or in some cases orthotopic. Representative cell types include tissue specific cells, progenitor cells, and stem cells. Matrices can be formed of synthetic or natural materials, by chemical coupling at the time of implantation, using standard techniques for formation of fibrous matrices from polymeric fibers, and using micromachining or microfabrication techniques. These devices and strategies are used as delivery systems via standard or minimally invasive implantation techniques for any number of parenterally deliverable recombinant proteins, particularly those that are difficult to produce in large amounts and/or active forms using conventional methods of purification, for the treatment of a variety of conditions that produce abnormal growth, including treatment of malignant and benign neoplasias, vascular malformations (hemangiomas), inflammatory conditions, keloid formation, abdominal or plural adhesions, endometriosis, congenital or endocrine abnormalities, and other conditions that can produce abnormal growth such as infection. Efficacy of treatment with the therapeutic biologicals is detected by determining specific criteria, for example, cessation of cell proliferation, regression of abnormal tissue, or cell death, or expression of genes or proteins reflecting the above.

[0001] This application claims priority to U.S. Ser. No. 60/178,842filed Jan. 27, 2000.

[0002] The United States government has certain rights in this inventionby virtue of Grant No. CA17393 from the National Institutes of Health toPatricia K. Donahoe and David T. MacLaughlin; National Institute ofHealth grant No. CA71345; and Department of Defense 1200-202487 toJoseph P. Vacanti.

BACKGROUND OF THE INVENTION

[0003] The present invention is generally in the area of methods andsystems for treatment of disorders such as cancer with biologicallyactive agents produced naturally by cells in extremely small quantities,using genetically engineered host cells or natural cells that secrete asubstance naturally implanted in biodegradable polymeric matrices.

[0004] One of the difficulties in treatment of conditions such as cancerusing protein or other biological modifiers is the need for largequantities of the therapeutic agent to be delivered over an extendedperiod of time. For most of the compounds discovered during research oncomplex pathways or unique tissues, it has not been possible, or has notbeen commercially feasible, to produce the compounds in sufficientquantity to treat the disorders. Numerous examples of these compounds,especially proteins, have been reported. One prominant example isangiostatin, a naturally occurring anti-angiogenic peptide identified byresearchers at Children's Medical Center in Boston, Mass. Althoughextremely promising in mice (O'Reilly et al., Science 285(5435):1926-81999), the inability of the developers to produce large quantities ofthe peptide has proven to be a major stumbling block to conductingclinical trials for treatment of cancer.

[0005] Mullerian Inhibiting Substance (MIS) is another biological withgreat potential for treatment of cancer. MIS is produced by the fetaltestis and causes the regression in males of the Müillerian duct, theforerunner of the female reproductive ducts. MIS has been shown to havegreat potential as a treatment for ovarian carcinomas (Chin et al,Cancer Research. 51:2101-2106, 1991; Masiakos, et al, Clinical CancerResearch, 5(11):3488-99 1999) which are derived from embryonic Müllerianstructures. Recombinant human MIS (rhMIS) produced in Chinese hamsterovary cells (CHO) in multiple roller bottles has antiproliferativeactivity against several human carcinoma cell lines (Chin, et al, 1991).Recently, it was also reported that rhMIS specifically binds to afunctional heteromeric serine threonine (Teixeira, et al., Androl.17(4):33641 1996; Teixeira et al., Endocrinology Jan;137(1):160-5 1996)receptor on the surface of human ovarian cancer ascites cells andinhibits the growth in vitro of these cells and of cells obtaineddirectly from women with Stage III and IV disease (Masiakos et al.,1999). See also, U.S. Pat. Nos. 4,404,199, 4,487,833, 4,510,131,4,753,794, 4,792,601, 5,011,687, 5,198,420 and 5,661,126 to Donahoe, etal., the teachings of which are incorporated by reference herein.

[0006] The Pediatric Surgical Research Laboratories has tested thehypothesis that MIS will be a useful therapeutic agent for certainepithelial ovarian cancers in a number of in vitro studies describedbelow, but only limited trials have been conducted in vivo. A majorobstacle has been purifying sufficient recombinant protein of suitablepotency and homogeneity for patient use.

[0007] Late stage epithelial ovarian cancer is a common and highlylethal gynecologic malignancy. Despite advances in treatment over thepast two decades substantial improvement in overall survival has beenslow and incremental and a high mortality remains. The coelomicepithelium, which invaginates to form the Müllerian duct, is also theorigin of these highly lethal human ovarian cancers. The hypothesis thatMIS is a therapeutic for these Müllerian derived tumors is predicated onprevious observations in which partially purified bovine MIS (Donahoe etal., J Surg Res. 23: 141-8, 1977) suppressed growth of a single humanovarian cancer cell line in monolayer culture (Donahoe et al., Science.205:913-5, 1979), in stem cell assays (Fuller et al., J Clin EndocrMetab. 54:1051-5, 1982), and in vivo in nude mice (Donahoe et al., AnnSurgery. 194:472-80,1981). Additionally, bovine MIS inhibited the growthof a large number of primary ovarian, Fallopian, and uterine carcinomasobtained directly from patients and tested in colony inhibition assaysin soft agar (Fuller et al., Gynecol. Oncol. 22:135-148, 1985). Afterpurifying bovine MIS (Budzik et. al., Cell 34: 307-314, 1983), thebovine and human MIS cDNAs and genomic human MIS (Cate et al., Cell. 45:685-98 1986) were cloned. The human gene was used to produce highlypurified recombinant human MIS (rhMIS) (Cate et. al., Cold Spring HarborSymp Quant Biol 51 Pt 1:641-7 1986; MacLaughlin et al., Methods Enzymol.198: 358-69, 1991) to which monoclonal and polyclonal antibodies wereraised for use in a sensitive ELISA (Hudson et al., J Clin EndocrinolMetab. 70: 16-22, 1990; Lee et al J Clin Endocrinol Metab. 81:571-69,1996). The rhMIS, which is now produced in a series of roller bottlesand purified from the media (Ragin et al, Protein Expression andPurification, 1992; 3(3):236-45), was shown to inhibit three humancarcinoma cell lines of Müllerian origin (Chin et al., 1991), as well asa human ocular melanoma cell line (Parry et al., Cancer Res. 52:1182-6,1992), in vitro and in vivo, in a dose dependent manner (Chin et al.,1991; Boveri et al., Int J Oncology. 2; 135-44, 1993). In order to scaleup production beyond the roller bottle capacity which suites academicneeds, a clonal line of MIS-producing transfected CHO cells (CHO, B9)was transferred to CHO B9 seeded bioreactors, for scale up to completethe phase I trials. Purification protocols for the bioreactor producedprotein have been designed (Ragin et al, 1992), but modifications toimprove purification protocols to enhance recovery and cioactivity havenot resulted in production of sufficient quantities.

[0008] It is important to note that like the other members of the TGFβfamily, the bioactive purified protein is not a single polypeptide chainbut a proteolytically cleaved molecule. MIS is primarily processed atresidue 427, producing 110 kDa amino-terminal and 25 kDa carboxyterminaldisulfide bond reduction sensitive homodimers (Pepinsky et al, J. Biol.Chem. 1988; 263:18961-4.; MacLaughlin et al, EndocrinologyJul;131(1):291-6 1992). Although the carboxy terminus is the activedomain of rhMIS in vitro, it has not been shown to be active in vivo.The non-covalent association of the amino and carboxy termini ispresumed to prevent the rapid clearance characteristic of the C terminusin vivo. Therefore, MIS to be administered to patients is being producedas a cleaved but non-dissociated complex. Unfortunately, theimmunoaffinity purification protocols result in aggregation, given thehydrophobic character of MIS, with a product of reduced potency and lowyield, making a process consistent with general manufacturing practicesproblematic. Moreover, alternative systems which are more efficient forlarge scale in vitro production, such as bacterial, yeast, or insectcell expression systems, have not been successful for the production ofbiologically active preparations of MIS.

[0009] It is therefore an object of the present invention to providemethods and reagents for production of clinically effective amounts oftherapeutic biologicals, especially proteins such as MIS, in vivo.

[0010] It is a further object of the present invention to providemethods and reagents for production of biologics in vivo, where theproduction can be discontinued if appropriate.

[0011] It is a still further object of the present invention to providemethods and reagents for treatment of a variety of disorderscharacterized by the proliferation of abnormal tissue, includingmalignant and benign neoplasias, vascular malformations, inflammatoryconditions including restenosis, infection, keloid formation andadhesions, congenital or endocrine abnormalities and other conditionsthat produce abnormal growth.

SUMMARY OF THE DISCLOSURE

[0012] Normal cells, such as fibroblasts or other tissue or organ celltypes, are genetically engineered to express biologically active,therapeutic agents, such as proteins that are normally produced in smallamounts, for example, MIS, Herceptin™, interferons, and Endostatin™, ornaturally produced compounds. These cells are seeded into a matrix forimplantation into the patient to be treated. Cells may also beengineered to include a lethal gene, so that implanted cells can bedestroyed once treatment is completed. Cells can be implanted in avariety of different matrices. In a preferred embodiment, these matricesare implantable and biodegradable over a period of time equal to or lessthan the expected period of treatment, during which the engrafted cellsform a functional tissue producing the desired biologically active agentfor longer periods of time. Representative cell types include tissuespecific cells, progenitor cells, and stem cells. Matrices can be formedof synthetic or natural materials, by chemical coupling at the time ofimplantation, using standard techniques for formation of fibrousmatrices from polymeric fibers, and using micromachining ormicrofabrication techniques.

[0013] These devices and strategies are used as delivery systems, whichmay be implanted by standard or minimally invasive implantationtechniques, for any number of parenterally deliverable recombinantproteins, particularly those that are difficult to produce in largeamounts and/or active forms using conventional methods of purification,for the treatment of a variety of conditions that produce abnormalgrowth, including treatment of malignant and benign neoplasias, vascularmalformations (hemangiomas), inflammatory conditions, keloid formationand adhesion, endometriosis, congenital or endocrine abnormalities, andother conditions that can produce abnormal growth such as infection.Efficacy of treatment with the therapeutic biologicals is detected bydetermining specific criteria, for example, cessation of cellproliferation, regression of abnormal tissue, or cell death.

[0014] The examples demonstrate the use of this method with a tissuespecific biological modifier, MIS. Genetically engineered CHO cells weregrown on implantable polymeric meshes and the levels of secreted rMISmeasured. The polymeric meshes with CHO cells seeded therein were thenimplanted in vivo and serum levels of rMIS measured. Data show very highlevels of rMIS over prolonged time periods. These animals were thenimplanted with human ovarian cell lines and tumor regression measured inthe presence of the MIS-producing cells. The implanted cellssignificantly inhibited the tumor cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a graph of MIS (ng/day) production over days ofincubation of CHO B9 cells impregnated on mesh in vitro.

[0016] FIGS. 2A-C are graphs of MIS production on polymeric implants inmice. FIG. 2A is a graph showing that the accumulation of MIS in serumis influenced by the size of the polymer implant. Polymer squares wereseeded with MIS-producing CHO-B9 cells 3 days prior to implantation.After 21 days, marked differences in the mean serum levels of MIS (n=4per polymer size) can be seen in the animals implanted with polymers ofvarying sizes. 0.5 cm² grafts were used for further study. FIG. 2B is agraph showing increasing levels of MIS were detected by ELISA on day 7,14, 21, and 28 in the serum of SCID mice after the CHO-B9 seeded polymerwas implanted in the right ovarian pedicle. This composite figure showsthe mean+/−the standard error of the mean for a total of 40 animals.FIG. 2C is a graph showing that the accumulation of MIS in the serum ofpolymer-bearing mice is reversible. Two weeks after implantation of apolymer-cell graft into the right ovarian pedicle of a SCID mouse theserum level of MIS was approximately 1200 ng/ml. Upon removal of thepolymer, the MIS falls to undetectable levels, consistent with lack ofmigration of MIS-producing cells from the site of implantation (n=1).

[0017]FIGS. 3A and 3B are graphs of MIS inhibition of the human ovariantumor cell line IGROV-1 tumor growth in animals implanted with CHO B9cells seeded on polyglycolic acid mesh as a function of graft size ratio(mean±SEM), compared to a mesh seeded with non-transfected L9 cells as anegative control. The graft-size ratio represents the size of the tumorat two weeks divided by the starting size of the tumor and represents ameasure of tumor growth. The L9 animals are represented by the squaresand the B9 animals by the diamonds. The dashed line indicates themean±the standard error. FIG. 3A shows that the mean graft-size ratios(GSR)+/−the standard error of the mean of the IGROV-1 tumors in theanimals producing bioactive MIS (n=8) was significantly smaller than theIGROV-1 tumors exposed to bioinactive MIS (n=10) (p value=0.016). A GSRof 1 indicates no net growth. FIG. 3B shows that the mean GSR of theIGROV-1 tumors implanted in the animals with MIS-producing CHO-B9polymer (n=30) was significantly smaller than the IGROV-1 tumorsimplanted in the animals with empty polymer (n=30) (p value<0.001).

DETAILED DESCRIPTION OF THE INVENTION

[0018] A strength of biological modifiers is that they impartspecificity to treatment paradigms to allow for prolonged parenteraltherapy, and eliminate many of the side effects and inconveniencesassociated with conventional therapies. Problems which are oftenencountered with these molecules include purification and enrichment.Most are manufactured in the laboratory using recombinant technology. Asmall number are selected for scale up by the pharmaceutical industry.As an important step for their purification, they undergo rigorouspurification schemes using separation methods that may alter theirchemical characteristics. As is often the case, the end product is asmall percentage of the starting material, and is frequently lesspotent. To obviate the loss of quantity and potency, a polymerscaffolding or matrix has been used to proliferate cells producing thebiological modifiers. When this scaffold or matrix is implanted into anorganism, it becomes vascularized or otherwise connected to thevasculature, the seeded cells grow to fill the scaffold, the biologicalmodifiers are secreted directly into the bloodstream or adjacent cells,and, in a preferred embodiment, the scaffold is resorbed, leaving a newsecretory tissue. Elimination of the purification steps enhances yieldand avoids the problems with contamination, cost and loss of biologicalactivity.

[0019] I. Materials for Production of Secretory Tissues

[0020] The materials required for production in vivo of biologicallyactive molecules include cells which produce the biologically activemolecules and matrices for proliferation of the engineered cells whichcan be implanted in vivo to form new secretory tissues. In oneembodiment, cells are obtained which already produce the desiredbiological modifiers. In another embodiment, cells are geneticallyengineered to produce the biological modifiers. In this embodiment, itis also necessary to provide the appropriate genes, means fortransfection of the cells, and means for expression of the genes.

A. Cells to be Engineered

[0021] As a proof of principle, CHO cells permanently transfected bycalcium phosphate precipitation with the MIS gene on a CMV promotor andclonally selected for the highest MIS producers were used inpreparations and implantations. The devices were seeded with cells for4-7 days prior to implantation and MIS levels measured in the serum by asensitive MIS ELISA. The next step was to implant non-tumor cells, suchas fibroblasts, both cell lines and then the patient's own fibroblaststo avoid rejection. These cells likewise can be engineered to express,and secrete the desired biological molecule(s). Other representativecell types include other patient specific differentiated cells,progenitor or embryonic or pluripotential stem cells.

[0022] Cells to be engineered can be obtained from established cellculture lines, by biopsy or from the patient or other individuals ofcompatible tissue types. The preferred cells are those obtained from thepatient to be treated. In those cases where the patient's own cells arenot used, the patient will also be treated with appropriateimmunosuppressants such as cyclosporine to avoid destruction of theimplanted cells during therapy.

[0023] In the preferred embodiments, cells are obtained directly fromthe donor, washed, and cultured using techniques known to those skilledin the art of tissue culture. Cells are then transfected with the geneof interest and seeded at various cell counts onto a matrix such as apolymeric mesh to achieve optimal production of a biological such asMIS.

[0024] Cell attachment and viability can be assessed using scanningelectron microscopy, histology, and quantitative assessment, forexample, by ELISA, fluorescent labelled or radioactive labelledantibodies. The function of the implanted cells can be determined usinga combination of the above-techniques and functional assays. Studiesusing protein assays can be performed to quantitate cell mass on thepolymer scaffolds. These studies of cell mass can then be correlatedwith cell functional studies to determine the appropriate cell mass.

B. Biologically Active Molecules

[0025] Any biologically active molecule which has been cloned or forwhich a cellular source is available can be used. Representativemolecules are those having a known activity which selectively reducesthe symptoms of the disorder to be treated, such as MIS, Herceptin,interferons, endostatin, and growth factors such as tumor necrosisfactor. For example, for the treatment of malignant or benignhyperplasia, the biologically active molecules include anti-angiogeniccompounds, MIS and other hormones which selectively or preferentiallybind to the cells to be killed or inactivated. Alternatively, the cellscan be engineered to correct the defect in the cells which results inoverproliferation.

[0026] The goal is not to form a permanent new tissue, but to provide animplanted “bioreactor” to produce therapeutic biologicals for a definedperiod effective to cause cessation of cell proliferation, regression ofabnormal tissue, or cell death. Various devices and strategies are usedas delivery systems which can be transplanted by standard or byminimally invasive implantation techniques for any number ofparenterally deliverable recombinant proteins, particularly those thatare difficult to produce in large amounts and/or active forms usingconventional methods of purification, for the treatment of a variety ofconditions that produce abnormal growth, including treatment ofmalignant and benign neoplasias, vascular malformations (hemangiomas),inflammatory conditions, keloid formation, endometriosis, congenital orendocrine abnormalities, and other conditions that can produce abnormalgrowth such as infection.

C. Vectors for Engineering Cells

[0027] Examples of recombinant DNA techniques include cloning,mutagenesis, and transformation. Recombinant DNA techniques aredisclosed in Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y. (1982). Vectors, including adeno-associatedviruses, adenoviruses, retroviruses, and tissue specific vectors, arecommercially available. Vectors can include secretory sequences, so thatthe biological modifier will diffuse out of the cell in which it isexpressed and into the vascular supply or interstitial spaces in orderto expose the cells of interest to concentrations of the protein thatare effective to treat the patient. The vector or expression vehicle,and in particular the sites chosen therein for insertion of the selectedDNA fragment and the expression control sequence, are determined by avariety of factors, e.g., number of sites susceptible to cleavage by aparticular restriction enzyme, size of the protein to be expressed,expression characteristics such as start and stop codons relative to thevector sequences, and other factors recognized by those of skill in theart. The choice of a vector, expression control sequence, and insertionsite for DNA sequence encoding the biological modifier is determined bya balance of these factors.

[0028] It should be understood that the DNA sequences coding for thebiological modifier that are inserted at the selected site of a cloningor expression vehicle may include nucleotides which are not part of theactual gene coding for the biological modifier or may include only afragment of the actual gene. It is only required that whatever DNAsequence is employed, a transformed host cell will produce thebiological modifier. For example, MIS DNA sequences may be fused in thesame reading frame in an expression vector with at least a portion of aDNA sequence coding for at least one eukaryotic or prokaryotic signalsequence, or combinations thereof. Such constructions enable theproduction of, for example, a methionyl or other peptidyl-MISpolypeptide. This N-terminal methionine or peptide may either then becleaved intra- or extra-cellularly by a variety of known processes orthe MIS polypeptide with the methionine or peptide attached may be used,uncleaved.

[0029] The complete nucleotide and amino acid sequence for human andbovine MIS, and cloning and expression vehicles, are provided in U.S.Pat. No. 5,047,336 to Cate, et al. and Cate et al., Cell45:685-698(1986), and are also available using publicly available genedata bases and commercial suppliers.

D. Matrices

[0030] There are three basic types of matrices that can be used: devicesformed by micromachining, micromolding or other microfabricationtechniques, fibrous polymeric scaffolds, and hydrogels.

[0031] 1. Microfabricated Device Design and Manufacture

[0032] Preferred materials for making devices to be seeded with cellsare biodegradable polymers, although in some embodiments non-degradablematerials may be preferred or may be used as structural support or ascomponents of a device formed of biodegradable polymer. The polymercomposition can be selected both to determine the rate of degradation aswell as to optimize proliferation. Many biodegradable, biocompatiblepolymeric materials can be used to form the device, or guide channelswithin the device, including both natural and synthetic polymers, andcombinations thereof. Examples of natural polymers include proteins suchas collagen, collagen-glycosaminoglycan copolymers, polysaccharides suchas the celluloses (including derivatized celluloses such asmethylcelluloses), extracellular basement membrane matrices such asBiomatrix, and polyhydroxyalkanoates such as polyhydroxybutyrate (PHB)and polyhydroxybutyrate-co-valerate (PHBV) which are produced bybacterial fermentation processes. Synthetic polymers include polyesterssuch as polyhydroxyacids like polylactic acid (PLA), polyglycolic acid(P GA) and compolymers thereof (PLGA), some polyamides andpoly(meth)acrylates, and polyanhdyrides. Examples of non-degradablepolymers include ethylenevinylacetate (EVA), polycarbonates, and somepolyamides.

[0033] The surface morphology of the devices can affect cell growth.Bioactive materials may also be incorporated into the device or asustained release matrix within the device to promote cell viability orproliferation. These materials can be incorporated into the polymer at aloading designed to release by diffusion and/or degradation of thepolymer forming the device over a desired time period, ranging from daysto weeks. Alternatively, the bioactive substance may be incorporatedinto a matrix loaded into or adjacent to the device. These matrices maybe formed of the same materials as the device or may consist ofpolymeric materials incorporated within the tracts or channels, forexample, hydrogel matrices of the types described in the literature (forexample, Wells, et al., Exp. Neurol. (1997) 146(2):395-402; Chamberlain,et al., Biomaterials 1998 19(15):1393-1403; and Woerly, et al., (1999)J. Tissue Engineering 5(5):467-488) for use in promoting nerve growth.Examples of such materials include polyamide, methylcellulose,polyethyleneoxide block compolymers such as the Pluronics, especiallyF127 (BASF), collagen, and extracellular matrix (ECM) of the type soldas Biomatrix. Other useful materials include the polymer foams reportedby Hadlock, et al., Laryngoscope (1999) 109(9):1412-1416.

[0034] Microfabrication techniques include micromachining, solid freeform (SFF) techniques, and micromolding techniques, as well as othertechniques based on well-established methods used to make integratedcircuits, electronic packages and other microelectronic devices, havingdimensions as small as a few nanometers and which can be mass producedat low per-unit costs.

Micromachining Techniques

[0035] Micromachining techniques are described in the literature, forexample, by Rai-Choudhury, ed. Handbook of Microlithography,Micromaching & Microfabrication (SPIE Optical Engineering Press,Bellingham, Wash. 1997), the teachings of which are incorporated herein.The techniques can be used to form the device directly, or as discussedbelow, to form molds which are then used to form the devices.

[0036] Other microfabrication processes that may be used includelithography; etching techniques, such as wet chemical, dry, andphotoresist removal; thermal oxidation; film deposition, such asevaporation (filament, electron beam, flash, and shadowing and stepcoverage), sputtering, chemical vapor deposition (CVD), epitaxy (vaporphase, liquid phase, and molecular beam), electroplating, screenprinting, lamination, laser machining, and laser ablation (includingprojection ablation). See generally Jaeger, Introduction toMicroelectronic Fabrication (Addison-Wesley Publishing Co., ReadingMass. 1988); Runyan, et al., Semiconductor Integrated Circuit ProcessingTechnology (Addison-Wesley Publishing Co., Reading Mass. 1990);Proceedings of the IEEE Micro Electro Mechanical Systems Conference1987-1998; Rai-Choudhury, ed., Handbook of MicrolithograhyMicromachining & Microfabrication (SPIE Optical Engineering Press,Bellingham, Wash. 1997).

[0037] Deep plasma etching can be used to create structures withdiameters on the order of 0.1 μm or larger. In this process, anappropriate masking material is deposited onto a substrate and patternedinto dots having the diameter of the desired tracts or channels. Thewafer is then subjected to a carefully controlled plasma. Those regionsprotected by the metal mask remain and form the tracts.

[0038] Another method for forming devices including tracts or channelsis to use microfabrication techniques such as photolithography, plasmaetching, or laser ablation to make a mold form, transferring that moldform to other materials using standard mold transfer techniques, such asembossing or injection molding, and reproducing the shape of theoriginal mold form using the newly-created mold to yield the finaldevice. Alternatively, the creation of the mold form could be skippedand the mold could be microfabricated directly, which could then be usedto create the final device.

Micromolding Techniques

[0039] Another method of fabricating tracts or channels utilizesmicromold plating techniques. A photo-defined mold first is firstproduced, for example, by spin casting a thick layer, typically 150 μm,of an epoxy onto a substrate that has been coated with a thinsacrificial layer, typically about 10 to 50 nm. Arrays of cylindricalholes are then photolithographically defined through the epoxy layer,which typically is about 150 μm thick. (Despont, et al.,“High-Aspect-Ratio, Ultrathick, Negative-Tone Near-UV Photoresist forMEMS,” Proc. of IEEE 10^(th) Annual International Workshop on MEMS,Nagoya, Japan, pp. 518-522 (Jan. 26-30, 1997)). The diameter of thesecylindrical holes defines the outer diameter of the tracts. The uppersurface of the substrate, the sacrificial layer, is then partiallyremoved at the bottom of the cylindrical holes in the photoresist. Theexact method chosen depends on the choice of substrate. For example, theprocess has been successfully performed on silicon and glass substrates(in which the upper surface is etched using isotropic wet or dry etchingtechniques) and copper-clad printed wiring board substrates. In thelatter case, the copper laminate is selectively removed using wetetching. Then a seed layer, such as Ti/Cu/Ti (e.g., 30 nm/200 nm/30 nm),is conformally DC sputter-deposited onto the upper surface of the epoxymold and onto the sidewalls of the cylindrical holes. The seed layershould be electrically isolated from the substrate. Subsequently, one ormore electroplatable metals or alloys, such as Ni, NiFe, Au, Cu, or Ti,are electroplated onto the seed layer. The surrounding epoxy is thenremoved, leaving molds which each have an interior annular hole thatextends through the base metal supporting the tracts. The rate andduration of electroplating is controlled in order to define the wallthickness and inner diameter of the tracts.

[0040] The molds made as described above and injection moldingtechniques can be applied to form the tracts or channels in the molds(Weber, et al., “Micromolding—a powerful tool for the large scaleproduction of precise microstructures”, Proc. SPIE—International Soc.Optical Engineer. 2879, 156-167 (1996); Schift, et al., “Fabrication ofreplicated high precision insert elements for micro-optical bencharrangements” Proc. SPIE—International Soc. Optical Engineer. 3513,122-134 (1998). These micromolding techniques can provide relativelyless expensive replication, i.e. lower cost of mass production.

Solid Free Form Manufacturing Techniques

[0041] As defined herein, SFF refers to any manufacturing technique thatbuilds a complex three dimensional object as a series of two dimensionallayers. The SFF methods can be adapted for use with a variety ofpolymeric, inorganic, and composite materials to create structures withdefined compositions, strengths, and densities, using computer aideddesign (CAD).

[0042] Examples of SFF methods include stereo-lithography (SLA),selective laser sintering (SLS), ballistic particle manufacturing (BPM),fusion deposition modeling (FDM), and three dimensional printing (3DP).In a preferred embodiment, 3DP is used to precisely create channels andpores within a matrix to control subsequent cell growth andproliferation in the matrix of one or more cell types having a definedfunction, such as nerve cells.

[0043] The macrostructure and porous parameters can be manipulated bycontrolling printing parameters, the type of polymer and particle size,as well as the solvent and/or binder. Porosity of the matrix walls, aswell as the matrix per se, can be manipulated using SFF methods,especially 3DP. Structural elements that maintain the integrity of thedevices during erosion can also be incorporated. For example, to providesupport, the walls of the device can be filled with resorbable inorganicmaterial, which can further provide a source of mineral for theregenerating tissue. Most importantly, these features can be designedand tailored using computer assisted design (CAD) for individualpatients to individualize the fit of the device.

[0044] Three Dimensional Printing (3DP).

[0045] 3DP is described by Sachs, et al., “CAD-Casting: DirectFabrication of Ceramic Shells and Cores by Three Dimensional Printing”Manufacturing Review 5(2), 117-126 (1992) and U.S. Pat. No. 5,204,055 toSachs, et al., the teachings of which are incorporated herein. Suitabledevices include both those with a continuous jet stream print head and adrop-on-demand stream print head. A high speed printer of the continuoustype, for example, is the Dijit printer made and sold by Diconix, Inc.,of Dayton, Ohio, which has a line printing bar containing approximately1,500 jets which can deliver up to 60 million droplets per second in acontinuous fashion and can print at speeds up to 900 feet per minute.Both raster and vector apparatuses can be used. A raster apparatus iswhere the printhead goes back and forth across the bed with the jetturning on and off. This can have problems when the material is likelyto clog the jet upon settling. A vector apparatus is similar to an x-yprinter. Although potentially slower, the vector printer may yield amore uniform finish.

[0046] 3DP is used to create a solid object by ink-jet printing a binderinto selected areas of sequentially deposited layers of powder. Eachlayer is created by spreading a thin layer of powder over the surface ofa powder bed. The powder bed is supported by a piston which descendsupon powder spreading and printing of each layer (or, conversely, theink jets and spreader are raised after printing of each layer and thebed remains stationary). Instructions for each layer are deriveddirectly from a computer-aided design (CAD) representation of thecomponent. The area to be printed is obtained by computing the area ofintersection between the desired plane and the CAD representation of theobject. The individual sliced segments or layers are joined to form thethree dimensional structure. The unbound powder supports temporarilyunconnected portions of the component as the structure is built but isremoved after completion of printing.

[0047] As shown in U.S. Pat. No. 5,204,055, the 3DP apparatus includes apowder dispersion head which is driven reciprocally in a shuttle motionalong the length of the powder bed. A linear stepping motor assembly isused to move the powder distribution head and the binder depositionhead. The powdered material is dispensed in a confined region as thedispensing head is moved in discrete steps along the mold length to forma relatively loose layer having a typical thickness of about 100 to 200microns, for example. An ink-jet print head having a plurality ofink-jet dispensers is also driven by the stepping motor assembly in thesame reciprocal manner so as to follow the motion of the powder head andto selectively produce jets of a liquid binder material at selectedregions which represent the walls of each cavity, thereby causing thepowdered material at such regions to become bonded. The binder jets aredispensed along a line of the printhead which is moved in substantiallythe same manner as the dispensing head. Typical binder droplet sizes arebetween about 15 to 50 microns in diameter. The powder/binder layerforming process is repeated so as to build up the device layer by layer.While the layers become hardened or at least partially hardened as eachof the layers is laid down, once the desired final part configuration isachieved and the layering process is complete, in some applications itmay be desirable that the form and its contents be heated or cured at asuitably selected temperature to further promote binding of the powderparticles. In either case, whether or not further curing is required,the loose, unbonded powder particles are removed using a suitabletechnique, such as ultrasonic cleaning, to leave a finished device.Finer feature size is also achieved by printing polymer solutions ratherthan pure solvents.

[0048] Stereo-lithography (SLA) and Selective Laser Sintering (SLS).

[0049] SFF methods are particularly useful for their ability to controlcomposition and microstructure on a small scale for the construction ofthese medical devices. The SFF methods, in addition to 3DP, that can beutilized to some degree as described herein are stereo-lithography(SLA), selective laser sintering (SLS), ballistic particle manufacturing(BPM), and fusion deposition modeling (FDM).

[0050] Stereolithography is based on the use of a focused ultra-violet(UV) laser which is vector scanned over the top of a bath of aphotopolymerizable liquid polymer material. The UV laser causes the bathto polymerize where the laser beam strikes the surface of the bath,resulting in the creation of a first solid plastic layer at and justbelow the surface. The solid layer is then lowered into the bath and thelaser generated polymerization process is repeated for the generation ofthe next layer, and so on, until a plurality of superimposed layersforming the desired device is obtained. The most recently created layerin each case is always lowered to a position for the creation of thenext layer slightly below the surface of the liquid bath. A system forstereolithography is made and sold by 3D Systems, Inc., of Valencia,Calif., which is readily adaptable for use with biocompatible polymericmaterials.

[0051] SLS also uses a focused laser beam, but to sinter areas of aloosely compacted plastic powder, the powder being applied layer bylayer. In this method, a thin layer of powder is spread evenly onto aflat surface with a roller mechanism. The powder is then raster-scannedwith a high-power laser beam. The powder material that is struck by thelaser beam is fused, while the other areas of powder remain dissociated.Successive layers of powder are deposited and raster-scanned, one on topof another, until an entire part is complete. Each layer is sintereddeeply enough to bond it to the preceding layer. A suitable systemadaptable for use in making medical devices is available from DTMCorporation of Austin, Tex.

[0052] Ballistic Particle Manufacturing (BPM) and Fusion DepositionModeling (FDM)

[0053] BPM uses an ink-jet printing apparatus wherein an ink-jet streamof liquid polymer or polymer composite material is used to createthree-dimensional objects under computer control, similar to the way aninkjet printer produces two-dimensional graphic printing. The device isformed by printing successive cross-sections, one layer after another,to a target using a cold welding or rapid solidification technique,which causes bonding between the particles and the successive layers.This approach as applied to metal or metal composites has been proposedby Automated Dynamic Corporation of Troy, N.Y.

[0054] FDM employs an x-y plotter with a z motion to position anextrudable filament formed of a polymeric material, rendered fluid byheat or the presence of a solvent. A suitable system is available fromStratasys, Incorporated of Minneapolis, Minn.

[0055] Polymer Materials, Binders and Solvents for use in SSF Techniques

[0056] Depending on the processing method, the material forming thematrix may be in solution, as in the case of SLA, or in particle form,as in the case of SLS, BPM, FDM, and 3DP. In the preferred embodiment,the material is a polymer. In SLS, the polymer must bephotopolymerizable. In the other methods, the material is preferably inparticulate form and is solidified by application of heat, solvent, orbinder (adhesive). In the case of SLS and FDM, it is preferable toselect polymers having relatively low melting points, to avoid exposingincorporated bioactive agent to elevated temperatures.

[0057] A number of materials are commonly used to form a matrix. Unlessotherwise specified, the term “polymer” will be used to include any ofthe materials used to form the matrix, including polymers and monomerswhich can be polymerized or adhered to form an integral unit, as well asinorganic and organic materials, as discussed below. In a preferredembodiment the particles are formed of a polymer which can be dissolvedin an organic solvent and solidified by removal of the solvent, such asa synthetic thermoplastic polymer, for example, ethylene vinyl acetate,poly(anhydrides), polyorthoesters, polymers of lactic acid and glycolicacid and other α hydroxy acids, polyhydroxyalkanoates, andpolyphosphazenes, a protein polymer, for example, albumin or collagen,or a polysaccharide. The polymer can be non-biodegradable orbiodegradable, typically via hydrolysis or enzymatic cleavage. Examplesof non-polymeric materials which can be used to form a part of thedevice or matrix for drug delivery include organic and inorganicmaterials such as hydoxyapatite, calcium carbonate, buffering agents,and lactose, as well as other common excipients used in drugs, which aresolidified by application of adhesive or binder rather than solvent. Inthe case of polymers for use in making devices for cell attachment andgrowth, polymers are selected based on the ability of the polymer toelicit the appropriate biological response from cells, for example,attachment, migration, proliferation and gene expression.

[0058] Photopolymerizable, biocompatible water-soluble polymers includepolyethylene glycol tetraacrylate (Mw 18,500) which can bephotopolymerized with an argon laser under biologically compatibleconditions using an initiator such as triethanolamine,N-vinylpyrrolidone, and eosin Y. Similar photopolymerizable macromershaving a poly(ethylene glycol) central block, extended with hydrolyzableoligomers such as oligo(d,1-lactic acid) or oligo(glycolic acid) andterminated with acrylate groups, may be used.

[0059] Examples of biocompatible polymers with low melting temperaturesinclude polyethyleneglycol 400 (PEG) which melts at 4-8° C., PEG 600which melts at 20-25° C., and PEG 1500 which melts at 44-48° C. Anotherlow melting material is stearic acid, which melts at 70° C.

[0060] Other suitable polymers can be obtained by reference to ThePolymer Handbook, 3rd edition (Wiley, N.Y., 1989), the teachings ofwhich are incorporated herein.

[0061] A preferred material is a polyester in thepolylactide/polyglycolide family. These polymers have received a greatdeal of attention in the drug delivery and tissue regeneration areas fora number of reasons. They have been in use for over 20 years in surgicalsutures, are Food and Drug Administration (FDA)-approved and have a longand favorable clinical record. A wide range of physical properties anddegradation times can be achieved by varying the monomer ratios inlactide/glycolide copolymers: poly-L-lactic acid (PLLA) andpoly-glycolic acid (PGA) exhibit a high degree of crystallinity anddegrade relatively slowly, while copolymers of PLLA and PGA, PLGAs, areamorphous and rapidly degraded.

[0062] Solvents and/or binder are used in the preferred method, 3DP, aswell as SLA and BPM. The binder can be a solvent for the polymer and/orbioactive agent or an adhesive which binds the polymer particles.Solvents for most of the thermoplastic polymers are known, for example,methylene chloride or other organic solvents. Organic and aqueoussolvents for the protein and polysaccharide polymers are also known,although an aqueous solution, for example, containing a crosslinkingagent such as carbodiimide or glutaraldehyde, is preferred ifdenaturation of the protein is to be avoided. In some cases, however,binding is best achieved by denaturation of the protein.

[0063] The binder can be the same material as is used in conventionalpowder processing methods or may be designed to ultimately yield thesame binder through chemical or physical changes that take place in thepowder bed after printing, for example, as a result of heating,photopolymerization, or catalysis.

[0064] These methods and materials are further described inPCT/US96/09344 “Vascularized Tissue Regeneration Matrices Formed bySolid Free-Form Fabrication Methods” Massachusetts Institute ofTechnology and Children's Medical Center Corporation.

[0065] 2. Fibrous Scaffolds for Implantation

[0066] Fibrous scaffolding can be used to implant the cells, forexample, as described in U.S. Pat. No. 5,759,830 to Vacanti, et al. Thedesign and construction of the scaffolding is of primary importance. Thematrix should be a pliable, non-toxic, porous template for vascularingrowth. The pores should allow vascular ingrowth and the injection ofcells into the scaffold without damage to the cells or patient. Thescaffolds are generally characterized by interstitial spacing orinterconnected pores in the range of at least between approximately 100and 300 microns in diameter. The matrix should be shaped to maximizesurface area, to allow adequate diffusion of nutrients and growthfactors to the cells and to allow the ingrowth of new blood vessels andconnective tissue.

[0067] The same type of polymers can be used as in the Solid Free FormManufacturing techniques described above. In the preferred embodiment,the matrix is formed of a bioabsorbable, or biodegradable, syntheticpolymer such as a polyanhydride, polyorthoester, polyhydroxy acid suchas polylactic acid, polyglycolic acid, or a natural polymer likepolyalkanoates such as polyhydroxybutyrate and copolymers or blendsthereof. Proteins such as collagen can be used, but is not ascontrollable and is not preferred. These materials are all commerciallyavailable. Non-biodegradable polymers, including polymethacrylate andsilicon polymers, can be used, depending on the ultimate disposition ofthe growing cells.

[0068] In some embodiments, attachment of the cells to the polymer isenhanced by coating the polymers with compounds such as basementmembrane components, agar, agarose, gelatin, gum arabic, collagens typesI, II, III, IV, and V, fibronectin, laminin, glycosaminoglycans,mixtures thereof, and other materials, especially attachment peptidesand polymers having attachment peptides or other cell surface ligandsbound thereto, known to those skilled in the art of cell culture.Vitrogen—100 collagen (PCO 701) has been used in these experiments.

[0069] 3. Hydrogel Matrices for Implantation

[0070] Polymeric materials which are capable of forming a hydrogel canbe utilized. The polymer is mixed with cells for implantation into thebody and is permitted to crosslink to form a hydrogel matrix containingthe cells either before or after implantation in the body. In oneembodiment, the polymer forms a hydrogel within the body upon contactwith a crosslinking agent. A hydrogel is defined as a substance formedwhen an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.Naturally occurring and synthetic hydrogel forming polymers, polymermixtures and copolymers may be utilized as hydrogel precursors. See forexample, U.S. Pat. No. 5,709,854 and WO 94/25080 by Reprogenesis.

[0071] In one embodiment, calcium alginate and certain other polymersthat can form ionic hydrogels which are malleable. For example, ahydrogel can be produced by cross-linking the anionic salt of alginicacid, a carbohydrate polymer isolated from seaweed, with calciumcations, whose strength increases with either increasing concentrationsof calcium ions or alginate. The alginate solution is mixed with thecells to be implanted to form an alginate suspension which is injecteddirectly into a patient prior to hardening of the suspension. Thesuspension then hardens over a short period of time due to the presencein vivo of physiological concentrations of calcium ions. Modifiedalginate derivatives, for example, more rapidly degradable or which arederivatized with hydrophobic, water-labile chains, e.g., oligomers ofε-caprolactone, may be synthesized which have an improved ability toform hydrogels. Additionally, polysaccharides which gel by exposure tomonovalent cations, including bacterial polysaccharides, such as gellangum, and plant polysaccharides, such as carrageenans, may be crosslinkedto form a hydrogel using methods analogous to those available for thecrosslinking of alginates described above. Additional examples ofmaterials which can be used to form a hydrogel include polyphosphazinesand polyacrylates, which are crosslinked ionically, or block copolymerssuch as Pluronics™ or Tetronics™, polyethylene oxide-polypropyleneglycol block copolymers which are crosslinked by temperature or pH,respectively. Other materials include proteins such as fibrin (althoughthis is not preferred since thrombin may stimulate tumor growth via apathway that MIS may have to overcome, such as EGF-stimulatedproliferation), polymers such as polyvinylpyrrolidone, hyaluronic acidand collagen. Polymers such as polysaccharides that are very viscousliquids or are thixotropic, and form a gel over time by the slowevolution of structure, are also useful. For example, hyaluronic acid,which forms an injectable gel with a consistency like a hair gel, may beutilized. Modified hyaluronic acid derivatives are particularly useful.Polymer mixtures also may be utilized. For example, a mixture ofpolyethylene oxide and polyacrylic acid which gels by hydrogen bondingupon mixing may be utilized. In one embodiment, a mixture of a 5% w/wsolution of polyacrylic acid with a 5% w/w polyethylene oxide(polyethylene glycol, polyoxyethylene) 100,000 can be combined to form agel over the course of time, e.g., as quickly as within a few seconds.

[0072] Covalently crosslinkable hydrogel precursors also are useful. Forexample, a water soluble polyamine, such as chitosan, can becross-linked with a water soluble diisothiocyanate, such as polyethyleneglycol diisothiocyanate. The isothiocyanates will react with the aminesto form a chemically crosslinked gel. Aldehyde reactions with amines,e.g., with polyethylene glycol dialdehyde also may be utilized. Ahydroxylated water soluble polymer also may be utilized.

[0073] Alternatively, polymers may be utilized which includesubstituents which are crosslinked by a radical reaction upon contactwith a radical initiator. For example, polymers including ethylenicallyunsaturated groups which can be photochemically crosslinked may beutilized, as disclosed in WO 93/17669. Additionally, water solublepolymers which include cinnamoyl groups which may be photochemicallycrosslinked may be utilized, as disclosed in Matsuda et al.,ASAIDTrans., 38:154-157 (1992).

[0074] II. Methods for Engineering and Implantation of Cells

[0075] A. Disorders to be Treated

[0076] A variety of conditions that produce abnormal growth, includingtreatment of malignant and benign neoplasias, vascular malformations(hemangiomas), inflammatory conditions including those resulting frominfection, especially chronic or recalcitrant conditions such as thosein the sinuses or which are cystic, keloid formation, endometriosis,congenital or endocrine abnormalities such as testotoxicosis (Teixeiraet al, PNAS 1999) and other conditions that produce abnormal growth, canbe treated.

[0077] Examples of tumor cells that can be treated with MIS includeprimary and metastatic growth of the following: ovarian adenocarcinomas,endometrial adenocarcinomas, cervical carcinomas, vulvar epidermoidcarcinomas, ocular melanomas, prostate, breast, and germ cell tumors. Asinitially demonstrated with MIS transfected cells, this methodology canbe used for delivery of a large number of proteins to control abnormaltissue growth, particularly other members of the TGFP family. Coupledwith minimally invasive delivery systems, the biodegradable implantsproducing the therapeutic proteins from transfected autologous cells canbe introduced into a variety of sites to deliver therapeutics,particularly where a local effect is advantageous. This allows use of avariety of recombinant proteins without the need for complexpurification protocols.

[0078] B. Engineering of Cells

[0079] In the preferred embodiment, patient cells are transfected withthe gene to be expressed, for example, rhMIS cDNA, to produce cellshaving stably incorporated therein the DNA encoding the molecules to beexpressed. Methods yielding transient expression, such as mostadenoviral vectors, are not preferred. Stable transfectants are obtainedby culturing and selection for expression of the encoded molecule(s).Those cells that exhibit stable expression are seeded onto/into theappropriate matrix and then implanted using techniques such as thosedescribed in the following examples.

[0080] C. Seeding of Matrices

[0081] The level of expression of the bioactive molecules is measuredprior to implantation to insure that an adequate number of cells isimplanted. In general, the higher the number of cells implanted, thebetter. Cells are preferably cultured initially in vitro, then implantedbefore the matrix degrades but when the level of bioactive molecules ishighest. An example of a suitable seeding density is between 1 and10×10⁶ cells on a matrix with a surface area of 0.25 cm².

[0082] D. Implantation of Matrices

[0083] The devices are implanted into the patient at the site in need oftreatment using standard surgical techniques. In one embodiment, thedevice is constructed, seeded with cells, and cultured in vitro prior toimplantation. The cells are cultured in the device, tested for high MISproduction by ELISA, then implanted.

[0084] The technique described herein can be used for delivery of manydifferent cell types for different purposes. Other endocrine producingtransfectant cells can also be implanted. The matrix may be implanted inone or more different areas of the body to suit a particularapplication. Matrices with hepatocytes or other high oxygen organ cellsmay be implanted into the mesentery to insure a good blood supply. Sitesother than the mesentery for injection or implantation of cells includethe ovarian pedicle, subcutaneous tissue, retroperitoneum, properitonealspace, and intramuscular space. The use of ovarian pedicle for MISproducing implants cause ovary and fallopian tubes to adhere to implants(Kristjansen, et al., 1994).

[0085] The need for these additional procedures depends on theparticular clinical situation.

III. EXAMPLES

[0086] The present invention will be further understood by reference tothe following non-limiting examples.

Example 1 Production of MIS in CHO Cells In Vitro

[0087] Materials and Methods

[0088] Polyglycolic acid fibers (approximately 12 microns diameter)obtained from Albany International were cut into 0.5×0.5 cm squares.1×10 10⁶ CHO B9 cells are seeded onto the mesh by the static seedingmethod. After 4 hours, the mesh was transferred into a new dishcontaining 10 ml of fresh media. Media MIS concentration was measuredserially over seven days by ELISA. Another CHO cell line, CHO L9, whichis transfected with a mutated rhMIS gene that produces non-cleavablebioinactive protein, was placed on the mesh as a negative control forthe implant experiments. Samples were also placed in the organ cultureassay to determine MIS bioactivity.

[0089] Results

[0090] In preliminary in vitro experiments, CHO B9 cells seeded onto apolyglycolic acid matrix produced large (i.e. microgram) quantities ofbioactive MIS as determined by ELISA (Hudson et al., J Clin EndocrinolMetab. 70: 16-22, 1990) and by a standard organ culture bioassay(Donahoe et al., 1977) which detects regression of the Müllerian duct.The results are shown in FIG. 1. After eight days, more than 2000 ng MISwere produced.

Example 2 Regression of Tumors by MIS-producing Cells Seeded ontoPolymeric Matrices Implanted into Animals

[0091] As described in Example 1, in vitro CHO cells transfected withthe human MIS gene were seeded onto a polyglycolic acid matrix andproduced large quantities (micrograms) of bioactive MIS as determined byELISA and by a standard in vitro organ culture bioassay. A productionrate of 400 ng/day/device was determined, corresponding to a productionrate per cell of 3 pg/cell/day. By serial sampling it was determinedthat 7-8 days incubation produced optimal bioactive MIS production bythe mesh impregnated with the B9 clone (a cell line tranfected with thehuman MIS genomic sequence).

[0092] Studies were then undertaken to determine MIS production withthis model in vivo. The MIS producing matrices were implanted into theovarian pedicle of B and T cell deficient 6-week-old female SCID mice.Serum levels of MIS were measured to determine the rate of rise andduration of MIS production by the explants. Supraphysiologic levels ofMIS were detected in mouse sera within three days of implantation. Itwas determined that the amount of MIS produced depends on the size ofthe mesh implanted.

[0093] Several ovarian cancer cell lines were tested in vitro forinhibition by MIS. Human ovarian cancer cell lines (IGROV-1, OVCAR-8,OVCAR-5) were plated on soft agarose and colony counts were determinedas an assay end point. Significant inhibition (20-80%) of these ovariancancer cell lines by MIS was observed. Ovarian cancer cell lines thatwere responsive to MIS in vitro were then placed beneath the renalcapsules of SCID mice. These tumors grew two to four fold at two weeksafter implantation with IGROV-1 showing the best growth. This representsan animal model of tumor growth.

[0094] The inhibitory properties of the MIS produced on the mesh wasthen tested in vivo against the IGROV-1 tumor cell line. B9 and L9impregnated meshes were implanted in mice. The L9 cells producebio-inactive MIS and served as the negative control for the experiment.MIS produced by the B9 clones significantly inhibited the growth of thehuman ovarian cancer cell line in vivo, as follows.

Materials and Methods

[0095] Animals

[0096] Severe combined immunodeficient (SCID) female mice and athymicnude mice (6 weeks old, average weight 18-20 g) were obtained from andstudied in the Edwin L. Steele Laboratory, Massachusetts GeneralHospital, Boston, Mass. All animals were cared for and experimentsperformed in this facility under AAALAS approved guidelines usingprotocols approved by the Institutional Review Board-InstitutionalAnimal Care and Use Committee of the Massachusetts General Hospitalprotocol #98-4254).

[0097] Cells

[0098] The IGROV-1 human ovarian cancer cell line was obtained from theAmerican Type Cell Culture and maintained in Dulbecco's Modification ofEagle's Medium (DMEM) supplemented with glutamine, penicillin,streptomycin, and 10% MIS-free female fetal calf serum. The cells weregrown at 37° C. in a humidified chamber perfused with 5% CO₂ in air. At80% confluency the cells were passed at a ratio of 1:4 and were carriedfor up to twelve passages.

[0099] The CHO-B9 cell line was formed by cloning the human MIS geneinto dihydrofolate reductase deficient Chinese hamster ovary cells asdescribed previously (Cate, et al. Cell, 45: 685-698 (1986)).Alternatively, a mutated cDNA which produces a bioinactive form of humanMIS was also cloned into the CHO cells, resulting in the CHO-L9 cellline (Kurian et al, Clin. Cancer Res. 1(3):343-349 (1995). Both celllines were maintained in DMEM supplemented with glutamine, penicillin,streptomycin, methotrexate, and 5% MIS-free female fetal calf serum.

[0100] Subrenal Capsule Assay

[0101] Following the method of Bogden et al. Exp Cell Biol 47(4):281-93(1979), as modified by Fingert, et al. Cancer Res., 47: 3824-3829(1987), the IGROV-1 tumor cell line was tested for growth in vivo in amurine subrenal capsule assay (Donahoe et al, 1984). Ten million cellswere centrifuged at 1500 rpm for 5 min to form a pellet. 300 microgramsof fibrinogen (Sigma; 20 mg/ml, dissolved in phosphate-buffered saline,pH 7.4) were added to the pellet, followed by 0.16 units of thrombin(Sigma; 20 unit/ml, dissolved in double-strength DMEM). This mixture wasincubated at 37° C. for 15 minutes. The cell clot thus formed was thencut into approximately 50 fragments (10 microliter volume, 200,000 cellseach) in preparation for implantation. Selected fragments were dissolvedwith trypsin and cells counted to confirm uniformity of cell number.

[0102] After inducing anesthesia using ketamine/xylazine (100/10 mg/kgBW, i.m.) a subcapsular space was developed in the left kidney with a19-gauge needle trocar and a cell clot measuring approximately 1×1ocular micrometer units at 7× magnification was introduced. The longestdiameter (L1) of the implant and the diameter perpendicular to thelongest diameter (W1) were measured with the ocular micrometer of adissecting microscope. The graft volume was estimated as L1×W1×W1. Theimplant was allowed to grow for 2-3 weeks at which time similarmeasurements were obtained at the same focal distance as the initialmeasurement to calculate the graft size ratio ([L2×W2×W2]/[L1×W1×W1]).Histology of the tumors was reviewed to verify that the implants wereviable and lacked both an inflammatory infiltrate and central necrosis.The growth of the tumor was measured at weekly intervals and the time ofthe experiment chosen so that 3 to 4× growth was achieved for thecontrol-treated tumors.

[0103] Preparation of the Polymer-cell Graft and its in vitro Productionof MIS

[0104] Biodegradable polymer consisting of 1 millimeter thick sheets ofnonwoven fibers of polyglycolic acid (density 70 mg/cc, fiber diameter14 μm, and average pore size 250 μm) was obtained from Smith and Nephew(York County, UK). The sheets were sectioned into 0.5 cm² squares whichwere placed in a 12-well tissue culture plate (Costar, Cambridge,Mass.), sterilized with 95% ethanol, and washed with phosphate buffersaline (PBS). Sterile filtered IN sodium hydroxide was added to eachwell for 60 seconds to make the polymer hydrophilic and the polymer waswashed with distilled water and coated with collagen (Vitrogen 3 mg/mldiluted 100× in sterile PBS) added to the wells for one hour at roomtemperature. The treated polymer squares were further washed withsterile PBS. Transfected CHO cells were remove from culture flasks withtrypsin-EDTA (Gibco) and resuspended in DMEM supplemental with 10%female fetal calf serum. After counting the cells in a Coulter counter,1-2×10⁶ suspended cells were seeded with a micropipet onto each polymersquare and absorbed onto the interstices of the polymer matrix during asubsequent incubation of 1-2 hours at 37° C. in 5% CO₂ air to allowsufficient time for attachment, after which time fresh media was addedand the wells returned to the incubator for attachment, after which timefresh media was added and the wells returned to the incubator for threeto seven days. Growth media was changed every other day. Theconcentration of MIS in the media was measured serially using asensitive MIS ELISA (Hudson et al, 1990) and an MIS specific organculture assay (Donahoe 1977) was used to assess MIS bioactivity onselected samples.

[0105] Implantation of Polymer-cell Graft and the IGROV-1 Tumor CellLine into SCID Mice

[0106] On day 0 the cells are seeded onto the polymer. 3-7 days later,when the media MIS levels reach 200 ng/ml, the polymer-cell graft isimplanted into the right ovarian pedicle of SCID mice. On day 10-14, theIGROV-1 tumor in the form of a cell clot is implanted under the leftrenal capsule of the mice. 2-3 weeks after implantation, the size of thetumors is measured. The left kidney and the right ovarian pedicle areremoved for immunohistochemical and/or histologic analysis. Serum iscollected throughout the protocol to determine MIS concentrations.

[0107] Three to seven days after seeding with CHO-B9 or CHO-L9 cells,when serum MIS was above physiologic levels, a 5×5×1 mm polymer squarewas implanted into the right ovarian pedicle of SCID mice as describedfor tumor samples by Kristjansen, et al. Microvasc. Res., 389-402(1994). After induction of anesthesia with ketamine/xylazine, a onecentimeter horizontal incision was made in the right flank. The ovarianpedicle was identified, delivered out of the wound, and the polymer-cellgraft laid on the ovarian pedicle and sutured in place with 6-0 prolene.Six to thirteen days later, the levels of circulating MIS weredetermined. When they approached supraphysiologic levels, the IGROV-1tumor cell line was prepared in a fibrin/thrombin cell clot andimplanted under the left renal capsule as described above. Differentsized polymers (0.125, 0.25, 0.5, and 1.0 cm²) seeded with CHO-B9 cellswere implanted into SCID mice and serum MIS levels measured to determinethe optimal size of the polymer implant. Two to three weeks afterimplantation of the IGROV-1 cell clot, the left kidney was exposed andthe dimensions of the implanted tumor measured. The graft size ratio wascalculated and comparisons made between groups of animals receiving theB9, L9, or empty polymer. Also, at this time, the right ovarian pedicle(location of polymer implantation) was removed and measured, andselected implants examined by immunohistochemistry or routine histology.The animals implanted with CHO-B9 seeded polymer served as theexperimental group and the animals implanted with CHO-L9 seeded or emptypolymer served as controls.

[0108] Serum MIS Levels and Bioassay

[0109] MIS was measured at various time points after polymerimplantation using a human MIS-specific ELISA described previously(Hudson et al, 1990). MIS-containing serum was placed in the MIS organculture assay (Donahoe et al, 1977) to correlate bioactivity of the MISpresent in the sample to the MIS levels as measured by ELISA.

[0110] Tissue Analysis

[0111] The tissue formed from the cell-polymer implant in the rightovarian pedicle and the kidneys with implanted IGOV-1 cell clot werefixed in 5% picric acid and 15% formalin in PBS. The tissue was thenprocessed and cut into 8 micron sections prior to staining either forroutine histologic analysis or for immunohistochemistry (Gustafson etal, N.Eng.J.Med. 326(7):466-471(1992). Viability of the tumors wasconfirmed as they were evaluated for central necrosis or an inflammatoryinfiltrate. Selected ovarian pedicles were harvested earlier during thecourse of the experiment and examined histologically to determine therate of biodegradation of the polymer.

[0112] Statistics

[0113] Values for the tumor graft-size ratio are expressed as mean+/−standard error (SE). An unpaired t-test performed by ‘STATVIEW’ andanalysis of variance (AVOVA) performed by ‘EXCEL’ were used to determinethe level of statistical significance (p values).

Results

[0114] Production of MIS in vivo

[0115] The polymer-cell graft was incubated in vitro for three to sevendays, at which time MIS levels of 100-400 ng/ml were measured in themedia and the graft was implanted into the ovarian pedicle of mice.Serum MIS was measured by ELISA in the animals implanted with thedifferent sized polymer squares (FIG. 2A). MIS levels in the animalsimplanted with polymer squares measuring 1.0 (n−4) and 0.5 (n=4) cm²were supraphysiologic at two weeks following implanation and exceeded 1microgram/ml 3 weeks after implantation. In the animals with smallersquares measuring 0.25 (n=4) and 0.125 (n=4) cm² the MIS levels weresupraphysiologic by three weeks following implantation. 0.5 cm² wasselected as the optimal size of seeded graft for implantation. The CHOcell line would not grow in animals made immunosuppressed byinactivation of the RAG-2 gene, hence the SCID mouse where growth andMIS production were robust was selected.

[0116] Serum MIS was measured by ELISA at 7, 14, 21, and 28 daysafter-implanatation of a polymercell graft measuring 0.5 cm² (n=40). At14 days, the levels of MIS measured 100-500 ng/ml and at 28 days thelevels measured 7-10 micrograms/ml (FIG. 2B). When the polymer-cellimplant was removed, the serum levels were undetectable within 7 days(n=1) (FIG. 2C).

[0117] Sera from mice with high MIS levels were analyzed in the MISbioassay to determine the bioactivity of the MIS produced by thepolymer-cell implant. The samples produced complete regression of theMullerian duct in the organ culture assay, indicating that the MIS inthe serum of animals retained bioactivity. The MIS produced in vivo isbioactive. When placed in an organ culture assay, serum from the animalswith a CHO-B9 polymer-cell graft implant causes complete regression ofthe rat Mullerian duct, leaving only the Wolffian duct. The negativecontrol culture shows both Mullerian and Wolffian ducts.

[0118] After two weeks in vivo, the polymer-cell graft formed a firm,living mass of tissue throughout the polymer fibers, which beganresorbing. After 4 weeks in vivo, the biodegradable polymer could nolonger be detected and the polymer-cell graft grew into a rounded,well-contained mass with an approximate diameter of 1.0 cm. There was noevidence of spread of CHO-B9 cells beyond the mass formed in the ovarianpedicle during the duration of the experiment. The mass consistedhistologically of epithelial cells with evidence of ingrowth of bloodvessels from the ovarian pedicle. Immunohistochemical analysis confirmedthe cells growing on the polymer continued to synthesize MIS.Immunohistochemistry of the polymer-cell graft after 4 weeks in vivoindicates ongoing production of MIS by the implanted cells. Thepolymer-cell graft stained with an antibody to human MIS is in markedcontrast to the staining pattern seen using a control antibody.

[0119] Inhibition of Tumor Growth by MIS Produced by Polymer-cellImplant

[0120] The IGROV-1 tumor cell line, when implanted into the subrenalcapsule of the SCID mice, formed measurable tumors growing to reach avolume graft-size ratio of 3-4 three weeks after implantation.Histologic analysis of these tumors demonstrated well-formed growthswith neovascularization from the underlying kidney parenchyma. There wasminimal necrosis and inflammatory infiltrate. After 3 weeks of exposureto MIS, the tumor is approximately one third the size of the control andalso has minimal necrosis or inflammation. When the IGROV-1 tumor cellline was implanted under the renal capsule of nude athymic mice, thetumors failed to achieve 3-4 fold growth three weeks after implantation.Hence the SCID mice were selected for use in subsequent experiments.

[0121] Animals implanted with the bioactive MIS-producing CHO-B9 polymer(n=8) showed very little net growth of the IGROV-1 implant, achieving amean graft-size ratio+/−the standard error of the mean of 1.62+/−0.218(FIG. 3A). The tumors implanted into the bioinactive MIS-producingCHO-L9 polymer (n=10) achieved a mean gift-size ratio of 3.25+/−0.506.This difference was statistically significant with a p-value of 0.016(FIG. 3A).

[0122] Three experiments were performed in which animals were implantedwith the bioactive MIS-producing CHO-B9 polymer or polymer withoutcells. In the first experiment the graft-size-ratios of IGROV-1 tumorswere 1.323+/−0.168 (n=10) for the animals with CHO-B9 polymer and3.427+/=0.682 (n=10) for the animals with polymer alone. In the secondexperiment the graft-size-ratio of the tumors in animals implanted withCHO-B9 polymer and polymer alone were 2.025+/−0.198 (n=10) and3.024+/−0.454 (n=10). In the third experiment the graft-size-ratio ofthe tumors in animals implanted with CHO-B9 polymer and polymer alonewere 1.427+/−0.147 (n=10) and 2.447+/−0.375 (n=10). The averagegraft-size ratios for all three experiments combined were 1.592+/−0.112in the CHO-B9 polymer animals and 2.966+/−0.299 in the animals withempty polymer (FIG. 3B). The difference in graft-size-ratio between thetwo groups was statistically significant with a p-value <0.001.

[0123] In summary, cells in this study were transfected with the geneencodinig human MIS, seeded onto biodegradable polyglycolic acid fibers,and shown to produce biologically active MIS in vitro. When thepolymer-cell grafts were implanted into animals, the continuallyproduced MIS could be detected in the serum of the animals within oneweek of implantation (FIG. 2A, B). Over time, secreted MIS was detectedby ELISA in increasing concentrations, and the serum, when tested in thestandard MIS in vitro bioassay, was biologically active (FIGS. 3A,B).There were no adverse effects in the animals as a result of the eitherthe presence of the polymer, the growing cells, or the high levels onMIS. Removal of the polymer-cell graft resulted in declining and, afterone week, undetectable levels of MIS (FIG. 2C) suggesting no spread ofcells from the ovarian pedicle. When tumor cell line responsive to MISin vitro was implanted in the subrenal capsule of the mice containingthe MIS secreting graft, the growth of the target tumor was slowedconsiderably. The graft-size ratio of the measured tumors wassignificantly smaller compared to the growth of tumors implanted inanimals with a polymer secreting biologically inactive MIS or a polymersecreting no MIS (FIGS. 3A, 3B). Thus only bioactive MIS and not a CHOcell product or biopolymer component was responsible for the growthinhibition. Histologic analysis confirmed the three-dimensional growthof the tumors and demonstrated lack of necrosis or excess inflammationthat could alter the size of the controls.

Example 3 rhMIS Production by Genetically Engineered, AutologousFibroblasts

[0124] To be used clinically, MIS must be administered to patients in asafe and cost effective manner in sufficient quantities to achieve tumorinhibition. MIS is a molecule that is produced by the Sertoli cells ofthe testis in the male and the granulosa cells of the ovary in thefemale. It serves the known function of Mullerian duct regression in thedeveloping male fetus and likely serves as a modifier of cell growth anddifferentiation in the male and female throughout life. MIS is a complexmolecule consisting of 70 kDa homodimer subunits that requires proteasemediated cleavage for biological activity; therefore, production anddelivery of purified MIS is predicted to be a complicated and costlyendeavor. As an alternative to production of the purified protein, theuse of biodegradable polymer seeded with MIS-producing transfected cellscan deliver biologically active MIS in quantities sufficient to achieveserum levels above the highest measured in newborn males, while avoidingcomplicated purification protocols.

[0125] The studies described herein demonstrate proof of principle forthe device using a partially transformed Chinese hamster ovaryepithelial cell line that can be tumorigenic in immunosuppressed mice.Normal human fibroblast cell lines and mouse fibroblasts harvested fromthe peritoneum of animals with rhMIS constructs have now beentransfected. Wild type and more easily cleavable MIS (S428R, Kurian etal., 1995) constructs will be transfected into fibroblasts using stateof the art stable transfection techniques optimized for transfectionefficiency and MIS production followed by clonal selection of the cellsthat produce the greatest concentration of MIS. These will be seededonto the biodegradable mesh and implanted into mice and experiments willbe repeated as with the CHO B9 cells. These experiments will be used toestablish the optimal geometry of the mesh that will produce the highestconcentration of MIS.

[0126] Human lung fibroblast cell lines IMR-90 will be permanentlytransfected with two monocistronic constructs encoding hygromycinresistance and one of either pCDNA-vector or pCDNA-K2, a CMV-driven MISligand expression construct. Transfection will be performed using eitherFugene 6 transfection reagent (Boehringer Mannheim) or using thestandard calcium phosphate DNA precipitation technique. Cells will beplated in 100 cm² well plates. When they reach 60 to 80% of confluence,Fugene 6 at 2 μg/ml will be added for 48 hours and washed. IMR-90 humanfibroblast cells have been transfected using the Fugene system with 0.5μg hygromycin and 5 μg of the K2 constructs, as well as vector alone,and these are now being selected in high concentration hygromycin media(750 μg/ml of hygromycin, Boehringer Mannheim). After two weeks themedia will be tested for MIS production by ELISA. Clones will bereplated at 10 cells/well in 24 well plates and expanded in mediacontaining 100 μg/ml of hygromycin. Clones will be selected for MISproduction by ELISA of overlying media; high producers will be grown onbiodegradable matrices and implanted in SCID mice harboring ovariantumors. Primary human fibroblasts originally taken from patients tostudy expression of androgen receptor will be similarly tested fortransfection efficiency using retroviral transfection and then clonallyselected for maximum production of MIS.

[0127] Since primary fibroblasts are difficult to transfect, anadenovirus transfection system adapted from one that used to transfectprimary Sertoli cells will be used. Briefly, 32.5 μl of adenovirus,diluted in PBS with 10% glycerol and 0.2% BSA, is added to the freshmedium and incubated for 1 hour at 37 C. After 1 hour of incubation,cells will be washed with HBSS and added in 500 μl of fresh medium. 48hours later cells the media will be collected and assayed for MIS usingthe MIS ELISA and the organ culture assay for regression of theMullerian Duct. The IMR-90 cells in 24 well plates have been transfectedwith a CMV-GFP virus and detected near 100% infection by fluorescence.Three other human fibroblast lines followed by primary fibroblasts willbe transfected, then virus to express the MIS construct generated.

[0128] Since growth after transfection and cloning may be inefficient infibroblast cell lines and primary fibroblasts, clones of cells may haveto be screened before choosing the best MIS producers to seed the mesh.An alternative approach is to grow pools of cells ex vivo on individualbiodegradable meshes and then to test and select each mesh for maximalproduction of MIS prior to implantation in vivo for serial measurementsof MIS in each animal's serum. Once the method of “cloning” in the meshor in monolayer culture is established, and the highest ex vivoproducers are selected, the loaded meshes will be implanted in anovarian fat pad and levels of MIS production measured and compared tothe levels produced by CHOB9 cell impregnated implants or by MIScontaining Alzet pumps.

[0129] The next step is to implant tumors in the subrenal capsule of 20SCID mice for as many days as it takes for the tumor to reach 4 timesthe original implant volume measured at length×width×width at the timeof implantation (Parry et al., 1992). The time to reach 4× for each cellline and the time to reach maximum production per unit number of cellsand unit size of mesh will be determined, then after the mesh isproducing high levels of MIS the tumors will be implanted. Growthbetween mesh containing MIS and non MIS producing fibroblasts (n=20) andAlzet delivered MIS positive controls (n=20) will then be compared.Subsequently tumor growth will be allowed to reach 3-4× in size. ThenMIS producing versus non-producing fibroblast-containing mesh will beimplanted intraperitoneally and growth of tumors compared between groupsto see if MIS prevents growth and/or reverses growth of the tumorimplants.

[0130] The biodegradable mesh impregnated with autologous fibroblastswill then be implanted into patients. It is important to note thateither dermal fibroblasts, peripheral or marrow stem cells, orperitoneal mesothelium will be requested from patients. The autologouscells will then be transfected and cloned and the optimal MIS producingfibroblasts will be impregnated in the biodegradable matrix plugs. Thematrices will then be implanted into the ovarian pedical in theperitoneal cavity of the ovarian cancer patient from whom thefibroblasts were taken. This may entail implantation in or near thetumors intraperitoneally for the ovarian cancer model or in the liver,brain, heart, blood vessels, joints, or other organs, as the protein ofinterest or therapeutic indication dictates.

[0131] This ongoing work uses transfected fibroblasts which growrobustly on the polymer. To avoid immunosuppression in patients, asample of the patient's own cells will be transfected with the human MISgene sequence. The cells could be fibroblasts or myofibroblasts obtainedfrom a small skin or muscle biopsy or stem cells from peripheral bloodor bone marrow. The cells would be grown on biodegradable polymer invitro and implanted in the patient, providing continual production ofMIS to serve as an inhibitor of tumor growth.

[0132] Modifications and variations of the present invention areintended to come within the scope of the following claims.

We claim:
 1. A method for treating a disorder characterized by excessiveproliferation of tissue comprising implanting a cell-matrix structurecomprising a matrix having attached thereto an effective amount of cellsstably expressing a gene encoding at least one biological modifier tostop or regress the excessive tissue proliferation, wherein the cellsare either genetically engineered to produce the biological modifier orof a different cell type than the tissue that has proliferatedexcessively.
 2. The method of claim 1 wherein the disorder is selectedfrom the group consisting of malignant and benign neoplasias, vascular,inflammatory conditions causing excessive proliferation of cells, keloidformation, intraperitoneal or intrathoracic adhesions, endometriosis,congenital or endocrine abnormalities, and infections causing excessiveproliferation of cells.
 3. The method of claim 1 wherein the matrix isselected from the group consisting of fibrous scaffolds, polymerichydrogels, and micromachine or micromolded substrates.
 4. The method ofclaim 1 wherein the cells are selected from the group consisting oftissue specific cells, progenitor cells, and stem cells.
 5. The methodof claim 4 wherein the cells are genetically engineered to produce thebiological modifier.
 6. The method of claim 5 wherein the biologicalmodifier is a protein.
 7. The method of claim 1 wherein the biologicalmodifier is selected from the group consisting of angiogenesisinhibitors, MIS, Herceptin, interferons, TGF-beta factors, steroid ororphan receptors, chimeric transcription factors, antibodies andantisense.
 8. The method of claim 7 wherein the biological modifier isMIS and the cells are engineered to secrete biologically active MIS toproduce serum levels effective to stop tissue proliferation or regressexcessive tissue.
 9. The method of claim 8 wherein the cell-matrixstructure is implanted into a patient with a disorder selected from thegroup consisting of vulvar epidermoid carcinomas, cervical carcinomas,endometrial adenocarcinomas, ovarian adenocarcinomas, ocular melanomas,prostate, lymphoid, breast, cutaneous, and germ cell tumors.
 10. Themethod of claim 1 wherein the inflammatory disorder is restenosis andthe cells are not endothelial cells, or an endocrine disorder.
 11. Themethod of claim 1 wherein the cells are genetically engineered toexpress the biological modifier from recombinant DNA encoding thebiological modifier.
 12. The method of claim 1 wherein the cells areselected based on natural production of the biological modifier and thecell-matrix structure is implanted at a site where the biologicalmodifier can stop proliferation or cause tissue regression.
 13. Acell-matrix structure for implantation into a patient having attachedthereto an effective amount of cells stably expressing a gene encodingat least one biological modifier to stop or regress excessive tissueproliferation in a patient in need thereof, wherein the cells are eithergenetically engineered to produce the biological modifier or of adifferent cell type than the tissue that has proliferated excessively.14. The cell-matrix structure of claim 13 wherein the cells produce abiological modifier effective to treat a disorder selected from thegroup consisting of malignant and benign neoplasias, vascular,inflammatory conditions causing excessive proliferation of cells, keloidformation, endometriosis, congenital or endocrine abnormalities, andinfections causing excessive proliferation of cells.
 15. The cell-matrixstructure of claim 13 wherein the matrix is selected from the groupconsisting of fibrous scaffolds, polymeric hydrogels, and micromachineor micromolded substrates.
 16. The cell-matrix structure of claim 13wherein the cells are selected from the group consisting of tissuespecific cells, progenitor cells, and stem cells.
 17. The cell-matrixstructure of claim 13 wherein the cells are genetically engineered toproduce the biological modifier.
 18. The cell-matrix structure of claim13 wherein the biological modifier is a protein.
 19. The cell-matrixstructure of claim 13 wherein the biological modifier is selected fromthe group consisting of angiogenesis inhibitors, MIS, angiogenesisinhibitors, MIS, Herceptin, interferons, TGF-beta factors, steroid ororphan receptors, chimeric transcription factors, antibodies andantisense.
 20. The cell-matrix structure of claim 19 wherein thebiological modifier is MIS and the cells are engineered to secretebiologically active MIS to produce serum levels effective to stop tissueproliferation or regress excessive tissue.
 21. The cell-matrix structureof claim 20 wherein the cell-matrix structure is implanted into apatient with a disorder selected from the group consisting of vulvarepidermoid carcinomas, cervical carcinomas, endometrial adenocarcinomas,ovarian adenocarcinomas, ocular melanomas, prostate, lymphoid, breast,cutaneous, and germ cell tumors.
 22. The cell-matrix structure of claim13 wherein the cells are genetically engineered to express thebiological modifier from recombinant DNA encoding the biologicalmodifier.
 23. The cell-matrix structure of claim 13 wherein the cellsare selected based on natural production of the biological modifier andthe cells are implanted at a site where the biological modifier can stopproliferation or cause tissue regression.